The immune system is a complex network of organs, tissues and immune cells that distinguish between self and non-self in order to protect the body from foreign materials. If its function is impaired, homeostasis is disrupted, leading to the development of disease or even death. Therefore, methods that identify compounds with immunomodulatory activity are needed in order to assess the risk that accompanies exposure to a particular compound. The determination of immunosuppressive activity of compounds is currently based on animal models, but an increasing number of alternative in vitro and in silico methods is being developed to replace existing tests. To this end, an in vitro model has been developed as an alternative to the human whole blood cytokine release assay, which is limited by inter-individual differences due to the use of samples from different individuals. The developed model is based on the use of a coculture of THP-1 and Jurkat cell lines representing monocytes and T lymphocytes. Using known immunosuppressive compounds with different mechanisms of action, we evaluated the suitability of the developed model to determine the immunosuppressive activity of the compounds. First, the cytotoxic activity of selected compounds was assessed by the resazurin reduction assay. We identified the following compounds as cytotoxic: dasatinib at concentrations above 1 μM, imatinib at 50 μM concentration and torin 1 at concentrations above 100 nM. Next, coculture cells were exposed to 10 μM and 100 nM concentrations of the selected compounds. The maximum concentration at which most of the compounds do not have cytotoxic effects on coculture cells is 10 μM, while the 100 nM concentration represents the concentrations reached by the tested compounds under in vivo conditions. The cells were then activated with a combination of activators and the concentration of secreted cytokines IL-2, IL-8 and TNF-α was determined by ELISA. At a concentration of 10 μM, we were able to demonstrate the immunosuppressive activity of dasatinib, imatinib, tofacitinib, idelalisib, mirdametinib and torin 1. At a concentration of 100 nM, we were able to demonstrate the immunosuppressive activity of tofacitinib, mirdametinib, torin 1 and dasatinib. However, we were not able to demonstrate immunosuppressive activity of apremilast, bucladesine and sulfasalazine with the developed model. We concluded that the model is not suitable for evaluating compounds that act as inhibitors of phosphodiesterases and compounds that inhibit the secretion of the cytokines IL-2 and TNF-α from THP-1 cells. The cytokine IL-8 would be the most suitable for the evaluation of the immunosuppressive activity of the compounds, as it allows for the demonstration of immunosuppression and is secreted by both THP-1 and Jurkat cells, whereas IL-2 and TNF-α are secreted only by Jurkat cells, so we do not get a full insight into the potential immunosuppressive activity of the compound. In the future, it would be worthwhile to assess the performance of the model using compounds with mechanisms of action that have not yet been tested and to include other types of immune cells in the model to make it more reflective of the composition of whole blood.
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